Protective immunity to infectious pathogens requires the coordinated differentiation of CD8+ and CD4+ T cells. Increasingly powerful and systematic profiling of the transcriptional, epigenetic, and metabolic changes that occur during this differentiation has identified an immense catalog of genes and pathways that could play a role in regulating T cell differentiation in response to acute infection. The challenge is now to ascribe functions to each of the genes that have been identified as part of the broad reprogramming of cell state that occurs during CD8+ and CD4+ T cell differentiation. This proposal seeks to use loss-of-function Cas9/CRISPR-mediated genetic screens in vivo to systematically identify the critical genes that govern the differentiation of CD8+ and CD4+ T cells in response to acute infection. We have developed a genetic screening platform that uses pooled, in vivo CRISPR-mediated loss-of- function genetic screens to identify genes that positively or negatively regulate the development of effector and memory populations. We have already used this platform to conduct pooled libraries with sequencing-based deconvolution to test in parallel the function of over 100 genes in CD8+ T cells responding to acute infection. This proof-of-principle screen successfully recovered known positive and negative regulators of effector differentiation. Building on these initial studies, we now propose to extend this system to identify and validate regulators of T cell response to acute infection in mouse models and human cells in the following aims: Aim 1: Curate candidate regulators from scRNA-seq profiles of CD8+ and CD4+ T cells during acute infection using scRNA-seq profiles. We will use massively parallel single-cell RNA-seq to deeply profile antigen-specific CD8+ and CD4+ T cells during acute infection and use computation modeling (with Core B) to identify candidate regulators. From this rich dataset we will identify a set of potential regulatory molecules that will be screened in Aim 2. Aim 2: Perform pooled CRISPR screens to identify regulators of CD8+ and CD4+ fate and function. We will use pooled CRISPR/Cas9 mediated genetic screens to survey the function of thousands of genes in regulating differentiation of CD8+ and CD4+ T cells. We will screen using defined phenotypes that will uncover genes that: initiate and sustain effector and memory differentiation; guide to a memory fate commitment; govern lineage choice; or regulate the acquisition of specific patterns of cytokine secretion. Aim 3: Validate candidates that regulate CD8 and CD4 differentiation in mouse models and human T cells. To validate these candidate regulators we will use an extensive set of cellular immunological assays in vivo and in vitro to probe their function individually using genome editing or conventional knockouts where available. We will complement these studies with CRISPR/Cas9 mediated gene editing in primary CD8+ and CD4+ T cells from healthy human subjects to test whether their regulatory role is conserved in human T cells.